Anionic lipids and lipid nano-structures and methods of producing and using same

ABSTRACT

Anionic non-phospholipids, as well as lipid nanostructures formed therefrom, are disclosed herein. Also disclosed are methods of producing and using same.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Contract NumberEB005187 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

Not Applicable.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The presently disclosed and claimed invention relates generally tocompositions comprising encapsulation materials, and in particular, butnot by way of limitation, to compositions comprising anionic lipidswithout a phosphate group, and methods of producing and using same.

2. Description of the Background Art

Vesicles encapsulating useful substances in the internal aqueous phaseand their dispersions are an important technology in various fields suchas pharmaceuticals, perfumes, cosmetics and food stuffs. Examples ofwidely used lipids that constitute the membrane of the vesicle/liposomeinclude negatively charged (anionic) phospholipids. Anionicphospholipids have previously been used in liposomes for impartingsize-stability, enhancing encapsulation of pharmaceuticals and/or tomodulate pharmacokinetics and pharmacodynamics of liposomes. However,said anionic phospholipids have been shown to induce untoward reactionsin biological systems, and the resultant toxicity has been manifested bysevere side effects such as but not limited to, acute thrombocytopenia,complement activation, dysfunction of white blood cells and the like.

Safety and adequate availability of blood is still a major concern intransfusion medicine. The requirement of pre-transfusion processing,storage and cross-matching of blood are other factors that have givenimpetus to the search for safe, shelf-stable and efficacious oxygencarrying fluids. An oxygen carrying fluid mimicking red blood cells(RBCs), in efficacy and safety profile is the goal. An ideal oxygencarrier would be hemoglobin that is encapsulated and is supplementedwith the oxido-reductive system of RBC. One approach to compartmentalizehemoglobin is to encapsulate hemoglobin in liposomes [Awasthi, 2005;Phillips et al., 1999; Takaori et al., 1996; Usuba et al., 1994; Sakaiet al., 1993; Farmer et al., 1988]. This encapsulated product has beenvariably termed hemoglobin vesicles (HbV), neo-red cells (NRC) orliposome-encapsulated hemoglobin (LEH), and said product contains highlyconcentrated (>36 g/dl) purified hemoglobin within the phospholipidmembranes. Hemoglobin vesicles or liposome-encapsulated hemoglobin (LEH)mimics membrane enclosed cellular structure of red blood cells [Phillipset al., 1999; Sakai et al., 1996; Rudolph, 1995]. Compared to freemodified hemoglobin preparations, LEH is characterized by spatialisolation of hemoglobin by an oxygen permeable lipid layer thateliminates the toxicity associated with free modified or unmodifiedhemoglobin.

A major impediment in the development of LEH has been the lowencapsulation efficiency of hemoglobin inside the vesicles. To increasethe encapsulation of proteins inside liposomes, anionic lipids, such asdimyristoyl- and dipalmitoyl-phosphatidyl glycerol (DMPG and DPPG) areusually incorporated in the lipid composition [Drummond et al., 1999;Walde et al., 2001]. However, anionic liposomes rapidly interact withthe biological system subsequent to their opsonization with complementand other circulating proteins [Miller et al., 1998; Szebeni, 1998].Such an interaction has at least two acute consequences—a rapid uptakeby the reticuloendothelial system (RES), and toxic effects, such aspseudoallergy that is manifested as vasoconstriction, pulmonaryhypertension, dyspnea, drop in circulating platelets and leukocytes,etc. [Awasthi et al., 2007; Szebeni et al., 2000]. Since these reactionsare mostly dependent on lipid dose, the problem is more challenging whenhuge quantities of liposomes need to be administered, such as in the useof LEH as a resuscitative fluid in acute blood loss. It is a challengetherefore, to encapsulate maximum amounts of hemoglobin in the leastamount of lipid using anionic lipids and to keep the charge-associatedtoxicity in check.

Therefore, there is a need in the art for new and improved lipids andliposome encapsulation methods that increase encapsulation and stabilitywhile overcoming the untoward effects commonly seen with prior artencapsulation methods. It is to said lipid compositions and lipidnanostructures formed therefrom, as well as methods of producing andusing same, that the presently disclosed and claimed invention isdirected.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 illustrates a scheme for the synthesis of saturated CHHDA inaccordance with the presently disclosed and claimed invention.

FIG. 2 illustrates a synthesis scheme for DPTSA in accordance with thepresently disclosed and claimed invention.

FIG. 3 illustrates a synthesis scheme for DPTGA in accordance with thepresently disclosed and claimed invention.

FIG. 4 illustrates a synthesis scheme for CHEMS in accordance with thepresently disclosed and claimed invention.

FIG. 5 graphically illustrates differential scanning calorimetry ofCHHDA.

FIG. 6 illustrates the effect of CHHDA mol % on Hb encapsulation.

FIG. 7 contains scanning electron micrographs of LEH prepared with 28mol % of CHHDA.

FIG. 8 is a schematic illustration of LEH manufacturing.

FIG. 9 graphically illustrates the cytotoxicity of LEH in HUVEC (a) andRAW cells (b).

FIG. 10 demonstrates the effect of LEH on activation of platelets invitro. ADP (100 μM) induced platelet activation, as characterized by anincrease in fluorescence intensity within the CD62P-positive region.However, a similar incubation with LEH demonstrated no such plateletactivation.

FIG. 11 graphically depicts an evaluation of effectiveness of LEH oncerebral energy metabolism in a 40% hemorrhagic shock model in ratsusing proton and phosphorous magnetic resonance spectroscopy (¹H-MRS and³¹P-MRS).

DETAILED DESCRIPTION OF THE INVENTION

Before explaining at least one embodiment of the invention in detail byway of exemplary drawings, experimentation, results, and laboratoryprocedures, it is to be understood that the invention is not limited inits application to the details of construction and the arrangement ofthe components set forth in the following description or illustrated inthe drawings, experimentation and/or results. The invention is capableof other embodiments or of being practiced or carried out in variousways. As such, the language used herein is intended to be given thebroadest possible scope and meaning; and the embodiments are meant to beexemplary—not exhaustive. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

Unless otherwise defined herein, scientific and technical terms used inconnection with the presently disclosed and claimed invention shall havethe meanings that are commonly understood by those of ordinary skill inthe art. Further, unless otherwise required by context, singular termsshall include pluralities and plural terms shall include the singular.Generally, nomenclatures utilized in connection with, and techniques of,cell and tissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g., Sambrook etal. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989) and Coligan et al.Current Protocols in Immunology (Current Protocols, Wiley Interscience(1994)), which are incorporated herein by reference. The nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are those wellknown and commonly used in the art. Standard techniques are used forchemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring.

The term “effective amount” refers to an amount of a biologically activemolecule or conjugate or derivative thereof sufficient to exhibit adetectable therapeutic effect without undue adverse side effects (suchas toxicity, irritation and allergic response) commensurate with areasonable benefit/risk ratio when used in the manner of the invention.The therapeutic effect may include, for example but not by way oflimitation, inhibiting the growth of undesired tissue or malignantcells. The effective amount for a subject will depend upon the type ofsubject, the subject's size and health, the nature and severity of thecondition to be treated, the method of administration, the duration oftreatment, the nature of concurrent therapy (if any), the specificformulations employed, and the like. Thus, it is not possible to specifyan exact effective amount in advance. However, the effective amount fora given situation can be determined by one of ordinary skill in the artusing routine experimentation based on the information provided herein.

As used herein, the term “concurrent therapy” is used interchangeablywith the terms “combination therapy” and “adjunct therapy”, and will beunderstood to mean that the patient in need of treatment is treated orgiven another drug for the disease in conjunction with thepharmaceutical compositions of the presently disclosed and claimedinvention. This concurrent therapy can be sequential therapy where thepatient is treated first with one drug and then the other, or the twodrugs are given simultaneously.

The terms “administration” and “administering”, as used herein will beunderstood to include all routes of administration known in the art,including but not limited to, oral, topical, transdermal, parenteral,subcutaneous, intranasal, mucosal, intramuscular and intravenous routes,including both local and systemic applications. In addition, the methodsof administration may be designed to provide delayed or controlledrelease using formulation techniques which are well known in the art.

The term “pharmaceutically acceptable” refers to compounds andcompositions which are suitable for administration to humans and/oranimals without undue adverse side effects such as toxicity, irritationand/or allergic response commensurate with a reasonable benefit/riskratio.

By “biologically active” is meant the ability to modify thephysiological system of an organism. A molecule can be biologicallyactive through its own functionalities, or may be biologically activebased on its ability to activate or inhibit molecules having their ownbiological activity.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 percent of allmacromolecular species present in the composition, more preferably morethan about 85%, 90%, 95%, and 99%. Most preferably, the object speciesis purified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

The term “patient” as used herein includes human and veterinarysubjects. “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including human, domestic and farm animals,nonhuman primates, and any other animal that has mammary tissue.

The terms “treat”, “treating” and “treatment”, as used herein, will beunderstood to include both inhibition of tumor growth as well asinduction of tumor cell death.

Administering a therapeutically effective amount or prophylacticallyeffective amount is intended to provide a therapeutic benefit in thetreatment, prevention, or management of a disease and/or cancer. Thespecific amount that is therapeutically effective can be readilydetermined by the ordinary medical practitioner, and can vary dependingon factors known in the art, such as the type of disease/cancer, thepatient's history and age, the stage of disease/cancer, and theco-administration of other agents.

The terms “liposome”, “lipid nanostructure” and “vesicle” may be usedinterchangeably herein and will be understood to refer to an assembledstructure constructed of molecules such as lipids and/or proteins, forexample, not through covalent bonds but through interactions (such asbut not limited to, hydrophobic interactions, electrostatic interactionsand hydrogen bonds) acting between the molecules in an aqueous medium.

The terms “aqueous solution” and “aqueous medium” will be usedinterchangeably herein and will be understood to refer to water as wellas any kind of solution which is physiologically acceptable and solventin water.

The presently disclosed and claimed invention is related to compositionscomprising a lipid having the structure represented by the followinggeneral formula [1]:

wherein R is NH or O; R′ is at least one of a hydrogen (H), an alkylgroup (such as but not limited to, methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, hexadecyl), Na,Li, K, a metal, or a halogen; R″ is at least one of a —CH₂— group and a—CH₂CH₂— group; and n and x are each an 8-16 carbon chain that may besaturated or unsaturated, and that may or may not contain additionalfunctional groups. In one embodiment, the lipid is asymmetrical.

Examples of lipid compositions of the presently disclosed and claimedinvention include, but are not limited to, 2-carboxyheptadecanoylheptadecylamide (CHHDA); 1,4-dipalmitoyl-tartarate-2,3-disuccinic acid(DPTSA); 1,4-dipalmitoyl-tartarate-2,3-diglutaric acid (DPTGA);1,4-disteroyl-tartarate-2,3-disuccinic acid (DSTSA); and cholesterylhemisuccinate (CHEMS).

The presently disclosed and claimed invention is also related to methodsof producing said compositions comprising said lipids of the generalformula [Awasthi, 2005]. The methods include the steps of reacting asaturated or unsaturated lipid dicarboxylic anhydride with another lipidcontaining at least one functional group, including but not limited to,an amine or hydroxyl functional group. The reaction is allowed to occurfor appropriate duration of time in the presence or absence of solvents,such as but not limited to, pyridine or triethanolamine (tritheylamine).A general method of synthesizing such lipids is illustrated by thesynthesis scheme discussed in Example 1. However, said synthesis schemeis to be understood to be provided for purposes of example only and isnot to be construed as limiting.

The presently disclosed and claimed invention is further related to aliposome or other lipid nanostructure comprising the lipid compositionsdescribed herein above. Said liposome or other lipid nanostructure mayfurther include other lipids, such as but not limited to, phospholipids.Specific examples of other lipids that may be utilized in accordancewith the presently disclosed and claimed invention include, but are notlimited to, at least one phosphatidylcholine, such as but not limitedto, 1,2-disteroyl-sn-glycero-3-phosphatidylcholine (DSPC) anddipalmitoyl phosphatidylcholine (DPPC); at least onephosphoethanolamine, such as but not limited to,1,2-disteroyl-sn-glycero-3-phosphatidylethanolamine (DSPE); at least onephosphatidylglycerol, such as but not limited to,dimyristoylphosphatidyl glycerol (DMPG); at least one sterol lipid, suchas but not limited to, cholesterol; at least one vitamin, such as butnot limited to, vitamin E; and the like. In one embodiment, the anionicnon-phospholipid may comprise 1% to 30% of the total lipid present inthe liposome/lipid nanostructure. In another embodiment, anyphospholipid present in the liposome/lipid nanostructure will be in therange of from 30% to 99% of the total lipid present in theliposome/lipid nanostructure, so as to minimize any toxicity of theliposome/lipid nanostructure.

The liposome/lipid nanostructure may be provided with any particle sizethat will allow the liposome/lipid nanostructure to function inaccordance with the presently disclosed and claimed invention. In oneembodiment, the liposome/lipid nanostructure may be provided with aparticle size in a range of from about 50 nm to about 500 nm, such asbut not limited to, about 200 nm to about 300 nm; in addition, theliposome/lipid nanostructure may be provided with a volume averageparticle size in a range of from about 10 nm to about 5,000 nm.

The lipids and liposome/lipid nanostructures formed therefrom inaccordance with the presently disclosed and claimed invention haveseveral advantages of the prior art, including but not limited to, adecrease in toxicity as well as a decrease in expense. Negativephospholipids are toxic to cells at certain concentrations; because ofthe absence of the phosphate group from the compositions of thepresently disclosed and claimed invention, the lipid (and thus theliposome/lipid nanostructures formed therefrom) do not induce untowardeffects as commonly seen with liposomes containing anionic phospholipid.It is shown herein that the presence of the anionic non-phospholipid ofthe presently disclosed and claimed invention is not toxic to vascularendothelial cells nor to macrophages in culture, and that LEHpreparations formed therefrom do not activate platelets in vitro. Inaddition, the lipid compositions of the presently disclosed and claimedinvention are entirely synthetic and thus can be synthesized in largequantities using inexpensive raw materials and procedures. Further, thereplacement of anionic phospholipids commonly used in liposomeformulations with the anionic non-phospholipid compositions of thepresently disclosed and claimed invention will increase encapsulationand stability of the liposomal structures.

The liposome/lipid nanostructure may further comprise at least oneadditional moiety. Moieties that may be utilized in accordance with thepresently disclosed and claimed invention include, but are not limitedto: (1) a targeting moiety such as but not limited to peptides thattarget the GRP receptor, including but not limited to, bombesin-relatedpeptides; antibodies and antibody fragments; as well as small and largemolecule ligands of known receptors and antigens; (2) a coating moleculeattached to any phospholipids present in the liposome/lipidnanostructure to decrease the charge effect thereof, such as but notlimited to, polyethylene glycol (PEG); (3) a labeling moiety, such asbut not limited to, moieties that allow radiolabeling of the liposomestructure, including but not limited to, diethylenetriamine pentaaceticacid; and the like.

In addition, the liposome/lipid nanostructure may have one or moremolecules/agents encapsulated there within. Said molecules that may beincorporated within the liposome/lipid nanostructure include anymolecules to be delivered to a patient. Examples include, but are notlimited to, hemoglobin, therapeutic or diagnostic drugs and therapeuticpeptides and proteins (such as but not limited to, interferon-gamma(IFN-γ); a reductant, such as but not limited to, glutathione, cystein,and homocysteine; an antioxidative enzyme, such as but not limited to,catalase and superoxide dismutase; an oxygen-affinity modifier, such asbut not limited to, pyridoxal phosphate; other enzymes, such as but notlimited to, glucose oxidase, glutathione peroxidase; chelating agents,such as but not limited to, deferoxamine; and combinations andderivatives thereof.

The molecule/agent encapsulated within the liposome/lipid nanostructuremay also be a pharmaceutical agent, and encapsulation thereof willproduce a pharmaceutical composition comprising the pharmaceutical agentencapsulated within the liposome/lipid nanostructure described in detailherein above. The pharmaceutical agent may be any molecule, proteinand/or enzyme that provide a diagnostic or therapeutic effect to apatient to which the pharmaceutical composition is administered.Examples of pharmaceutical agents that may be utilized in accordancewith the presently disclosed and claimed invention include, but are notlimited to, antibiotic/antiviral/antifungal agents, such as but notlimited to, amphotericin B and vancomycin; chemotherapeutic agents, suchas but not limited to, doxorubicin; anti-inflammatory drugs, such as butnot limited to, thalidomide; curcumin or a derivative thereof;diagnostic agents, such as but not limited to, iodinated X-ray and CTcontrast agents; radiodiagnostic and radiotherapeutics, such as but notlimited to, radionuclides Re-188, Tc-99m, I-131 and other iodineisotopes, F-18, C-11, O-15, In-111; and the like.

The presently disclosed and claimed invention is yet further related tomethods of producing the liposome/lipid nanostructure described hereinabove. Such methods include the steps of providing at least one anionicnon-phospholipid composition as described herein above, disposing samein an aqueous solution, and dispersing the compositions to form theliposome/lipid nanostructure. The dispersion may be accomplished by anymethod that supplies a sufficient amount of energy to cause the lipidcompositions to disperse into individual vesicles/liposomes/lipidnanostructures; said methods include, but are not limited to,sonication, extrusion, reverse phase evaporation, lyophilization,fluidized-bed, freeze thaw method, a method using a microfluidizer, andthe like.

The presently disclosed and claimed invention further includes a methodof forming a liposome/lipid nanostructure having at least onemolecule/agent encapsulated therein, such as but not limited to, thepharmaceutical composition described herein above. Such method includesthe steps of providing at least one anionic non-phospholipid compositionas described herein above, providing the molecule/agent/pharmaceuticalagent, disposing the anionic non-phospholipid composition and themolecule/agent/pharmaceutical agent in an aqueous solution, anddispersing same to form the liposome/lipid nanostructure having themolecule/agent/pharmaceutical agent encapsulated therein. The dispersionmay be accomplished as described herein above. Alternatively, theanionic non-phospholipid composition may initially be dispersed to forma pro-liposome composition, and the pro-liposome composition mixed withthe molecule/agent/pharmaceutical agent to encapsulate same and form theliposome/lipid nanostructure having the molecule/agent/pharmaceuticalagent encapsulated therein.

The presently disclosed and claimed invention is also directed to amethod of using the pharmaceutical composition described herein above.Said method includes the steps of providing the pharmaceuticalcomposition comprising anionic non-phospholipid and pharmaceutical agentas described herein above, and administering an effective amount of thepharmaceutical composition to a patient in need thereof.

One non-limiting example of a pharmaceutical agent that may be utilizedin accordance with the presently disclosed and claimed invention is thenovel curcumin analog 3,5-bis(2-fluorobenzylidene)-4-piperidone (EF24).The compound was encapsulated inside the liposomes using thedehydration-rehydration method in the presence of EF24 solubilized inhydroxypropyl beta cyclodextrin (HPβCD). The use of cyclodextrin is notto be construed as limiting, as the cyclodextrin may be replaced withother solubilizing substances, such as but not limited to, gammacyclodextrin or various other derivatives of cyclodextrins. TheEF24-liposomes are prepared for application in diseases such as butlimited to cancer, inflammation and infection. A non-limiting example ofa method of preparation of EF24-liposomes is described in detail inExample 2; however, said method is strictly for purposes of illustrationonly and is not to be construed as limiting.

The presently disclosed and claimed invention is further directed to apharmaceutical composition/artificial oxygen carrier comprisingliposome-encapsulated hemoglobin (LEH), wherein the liposome comprisesthe lipid of the general formula [1] and formed as described hereinabove. In one embodiment, the artificial oxygen carrier may furtherinclude at least one of a reductant, antioxidative enzyme andoxygen-affinity modifier (each of which is described in detail hereinabove) encapsulated therein, to enhance the resuscitative capacity ofthe liposome-encapsulated hemoglobin.

The hemoglobin utilized in accordance with the presently disclosed andclaimed invention may be obtained from any source, including nativehemoglobin derived from various living organisms as well as recombinanthemoglobin. In one embodiment, hemoglobin may be obtained from outdatedred blood cell units. The hemoglobin may further be provided in a morestable form, such as but not limited to, carbonylhemoglobin.Alternatively, the presently disclosed and claimed invention alsoencompasses the use of any heme derivatives. The term “heme derivatives”as used herein will be understood to include any derivatives orcompounds in which a porphyrin ring of heme is modified with asubstituent and has a reversible oxygen-binding potential.

Hemoglobin may be present in the artificial oxygen carrier at anyconcentration sufficient to allow the LEH to function as an artificialoxygen carrier. In one embodiment, the concentration of hemoglobinpresent in the LEH is in a range of from about 5 g/dL to about 15 g/dL.In addition, in one embodiment, the particle size of the LEH is in arange of from about 200 nm to about 300 nm.

The presently disclosed and claimed invention is also directed to amethod of forming said artificial oxygen carrier comprisingliposome-encapsulated hemoglobin. Such method includes the steps ofproviding at least one anionic non-phospholipid composition as describedherein above, providing an effective amount of hemoglobin, disposing theanionic non-phospholipid composition and the hemoglobin in an aqueoussolution, and dispersing same to form the liposome having hemoglobinencapsulated therein. The dispersion may be accomplished as describedherein above. When the artificial oxygen carrier comprises at least oneof a reductant, antioxidative enzyme and oxygen-affinity modifier, saidcomposition(s) may be provided and disposed in the aqueous solutionprior to dispersal thereof to form the liposomal structures, or thecomposition(s) may be added to the liposomal structures after formationthereof. For example, the encapsulated material may be encapsulatedduring multilamellar liposome preparation wherein the dry lipid isexposed to the composition. Alternatively, the lipid may first bereduced into liposome form to provide proliposomes, followed by exposureof the proliposomes to the composition to be encapsulated therein.

The presently disclosed and claimed invention is further directed to amethod of using the artificial oxygen carrier/liposome-encapsulatedhemoglobin described herein above. Said method includes the step ofproviding the liposome-encapsulated hemoglobin and administering aneffective amount of the LEH to a patient in need thereof. In oneembodiment, the patient exhibits one or more of the followingconditions: acute blood loss, surgery, anemia, hypovolemia, ischemia; inaddition, LEH administration may be indicated to increase oxygen contentin blood or tissue for enhanced chemotherapeutic or radiotherapeuticeffect in diseases, such as but not limited to, cancer. LEH may also beused in situations where religious beliefs prohibit whole blood or RBCtransfusion practice.

Examples are provided hereinbelow. However, the present invention is tobe understood to not be limited in its application to the specificexperimentation, results and laboratory procedures. Rather, the Examplesare simply provided as one of various embodiments and is meant to beexemplary, not exhaustive.

Example 1

This example provides a general synthesis scheme for one non-limitingexample of anionic non-phospholipid constructed in accordance with thepresently disclosed and claimed invention.

Tetradecenylsuccinic anhydride (4.3 g, 17.8 mmole) and hexadecylamine(2.93 g, 10.4 mmol) were weighed in an oven-dried round bottom flask.Pyridine (4.5 ml) was added to the flask and the reaction was allowed tooccur at 80° C. for 3 hr. The reaction mixture was extracted intodichloromethane (DCM, 150 ml) and washed with 10% HCl. The organic phasewas separated, dried with sodium sulfate and concentrated to obtainwhite solid of unsaturated CHHDA (wt. 6.88 g, yield 96%). Theunsaturated CHHDA was subjected to catalytic reduction to obtainsaturated CHHDA. Briefly, unsaturated CHHDA was dissolved in hexane at60° C. Reduction was carried out by passing hydrogen gas in presence ofcatalyst palladium/charcoal (5%, 40 mg) at atmospheric pressure for 16hrs. The reaction mixture was filtered on a Buchner funnel and hexanewas evaporated under vacuum to obtain CHHDA as white solid (wt. 5.36 g,yield 78%). ¹H NMR (300 MHz, CDCl₃): δ 5.72 (br, 1H, NH, exchanged withD₂O), 3.25 (q, 2H, CH₂, J=3.6), 2.80-2.35 (m, 4H, CH₂), 1.60-1.40 (m,4H, CH₂), 1.35-1.15 (m, 50H, CH₂), 0.87 (t, 6H, CH₃). ¹³C NMR (75 MHz,CDCl₃): δ 176.04, 171.43, 41.62, 37.41, 31.56, 31.51, 29.34, 29.30,29.16, 29.00, 22.34, 13.85. ESI HRMS calculated for C₃₄H₆₈NO₃ (M⁺+1)538.42. Found 538.40. CHHDA was also evaluated by differential scanningcalorimetry (DSC) on a Q1000 (TA Instruments, New Castle, Del.).

Example 2

Encapsulation of the novel curcumin analog3,5-bis(2-fluorobenzylidene)-4-piperidone (EF24) in liposomesconstructed in accordance with the presently disclosed and claimedinvention.

Formation of inclusion complexes: Inclusion complexes of EF24 with HPβCDwere formed in the solution phase. EF24 (15 mg) was transferred to 2.5ml of HPβCD solution (500 mg/ml). The mixture was continuously agitatedon a shaker incubator at 25° C. for 72 hrs and then allowed to stand for6 hrs. The mixture was centrifuged at 14000 rpm for 15 min and thesupernatant collected was passed through 0.22 μm cellulose acetatesterile filter. The amount of HPβCD-solubilized EF24 was estimatedspectrophotometrically at 315 nm. The HPβCD-EF24 inclusion complex wascharacterized by X-ray diffraction and differential scanningcalorimetry.

Preparation of Dehydration-Rehydration Vesicles (Drvs): the Dehydrationrehydration vesicles were prepared using a lipid composition describedearlier. The phospholipid film was rehydrated with HYPURE™ endotoxinfree cell culture grade water (Hyclone, Utah) to maintain total lipidconcentration to 12 mM. The resulting suspension of multilamellarvesicles was either subjected to 8 FT cycles or sonication. An FT cycleincluded snap-freezing the suspension in liquid nitrogen, followed byimmediate thawing in a 58° C. water bath. In case of sonication, a bathsonicator (Model 150D, VWR International, PA) was used, and liposomalsuspension was sonicated for 45 min at 58° C. A 0.22 μm filteredsolution of EF24-HPβCD inclusion complex in HYPURE™ water was added tothe liposomal suspension in such a way that the DSPC:EF24 molar ratioremained either 1.43 or 4.73. The mixture was then vortexed for 30 secand diluted with either 1% sucrose solution or phosphate buffered saline(PBS, pH 7.4) to provide a lipid concentration of 2 mM. The mixture wasdistributed in several sterile glass vials and lyophilized for 48 hrs ina Triad lyophilizer (Labconco, Mo.). The dried mass was rehydrated in acontrolled manner, typical in dehydration rehydration procedures. Thethick suspension formed was allowed to stand at 25° C. for 30 min andfurther diluted by several fold with Dulbecco's 10×PBS. The liposomeswere purified from un-entrapped materials using a two stepcentrifugation process. The first step involved a low speedcentrifugation (1500 rpm, 5 min). This step was repeated 3 times, eachtime transferring the supernatant into a sterile ultracentrifuge tubeand washing the cake with 1×PBS. The low speed centrifugation removedfree EF-24. The second step involved ultracentrifugation of thesupernatant that was collected during the first step (35,000 rpm at 4°C. for 40 min). This step was also repeated three times as above. FreeHPβCD, if any present, was removed during this step. The cake formedafter final cycle was reconstituted to 400 μl volume with sterile PBS.Strict aseptic conditions were maintained during the entire experiment.

Example 3 Materials and Methods for Example 3

Materials: The chemicals were obtained from Sigma-Aldrich (St. Louis,Mo.) and/or various suppliers through VWR Scientific (West Chester, Pa.)and were used without further purification. Tetradecenyl succinicanhydride was a kind gift from Vertellus Specialties Inc (Indianapolis,Ind.). For liposome preparations, the phospholipids were purchased fromLipoid (Ludwigshafen, Germany), Avanti Polar Lipids (Alabaster, Ala.) orNOF Corporation, (Tokyo, Japan). Cholesterol was obtained fromCalbiochem (Gibbstown, N.J.). Outdated red blood cell units were kindgift from Oklahoma Blood Institute (Oklahoma City, Okla.). For NMR andmass spectroscopy, analytical services in the Chemistry Department ofthe University of Oklahoma (Norman, Okla.) were used.

Synthesis of Anionic Nonphospholipids

2-Carboxyheptadecanoyl heptadecylamide (CHHDA, FIG. 1): The scheme forthe synthesis of CHHDA is shown in FIG. 1. Tetradecenylsuccinicanhydride (4.3 g, 17.8 mmole) and hexadecylamine (2.93 g, 10.4 mmol)were weighed in an oven-dried round bottom flask. Pyridine (4.5 ml) wasadded to the flask and the reaction was allowed to occur at 80° C. for 3hours. The reaction mixture was extracted into dichloromethane (DCM, 150ml) and washed with 10% HCl. The organic phase was separated, dried withsodium sulfate and concentrated to obtain white solid of unsaturatedCHHDA (wt. 6.88 g, yield 96%). The unsaturated CHHDA was subjected tocatalytic reduction to obtain saturated CHHDA. Briefly, unsaturatedCHHDA was dissolved in hexane at 60° C. Reduction was carried out bypassing hydrogen gas in presence of catalyst palladium/charcoal (5%, 40mg) at atmospheric pressure for 16 hours. The reaction mixture wasfiltered on a Buchner funnel and hexane was evaporated under vacuum toobtain CHHDA as white solid (wt. 5.36 g, yield 78%). ¹H NMR (300 MHz,CDCl₃): δ 5.72 (br, 1H, NH, exchanged with D₂O), 3.25 (q, 2H, CH₂,J=3.6), 2.80-2.35 (m, 4H, CH₂), 1.60-1.40 (m, 4H, CH₂), 1.35-1.15 (m,50H, CH₂), 0.87 (t, 6H, CH₃). ¹³C NMR (75 MHz, CDCl₃): δ 176.04, 171.43,41.62, 37.41, 31.56, 31.51, 29.34, 29.30, 29.16, 29.00, 22.34, 13.85.ESI HRMS calculated for C₃₄H₆₈NO₃ (M⁺+1) 538.42. Found 538.40. CHHDA wasalso evaluated by differential scanning calorimetry (DSC) on a Q1000 (TAInstruments, New Castle, Del.). DSC was performed, courtesy Dr. Brian P.Grady, in the School of Chemical, Biological and Materials Engineeringof the University of Oklahoma (Norman, Okla.).

1,4-Dipalmitoyl-tartarate-2,3-disuccinic acid (DPTSA, FIG. 2): Cetylalcohol (71 gm, 0.29 mol) and p-toluene sulfonic acid (PTSA, 30.4 gm,0.16 mol) were added to a solution of tartaric acid (20 gm, 0.13 mol) intoluene. The reaction mixture was refluxed for 16 h using dean starkapparatus. The toluene layer was given a water wash (100 ml), and theorganic phase was separated, dried over sodium sulfate and concentratedto obtain white solid of DPTA. The solid was recrystallized from DCM andhexane to get 1,4-dipalmitoyl tartarate (DPTA) as white crystallinesolid (75 gm, 86% yield). The melting point was determined to be 40-41°C. ¹H NMR (300 MHz, CDCl₃): δ 4.52 (s, 2H), 4.25 (t, 4H, CH₂), 1.75-1.60(m, 4H, CH₂), 1.40-1.10 (m, 52H, CH₂), 0.87 (t, 6H, CH₃). Disuccinateester of DPTA was synthesized by adding succinic anhydride (0.91 gm, 9.1mmol) to a solution of DPTA (2 gm, 3.0 mmol) in pyridine (5 ml) andheating at 80° C. for 2 h. The resultant compound was extracted intochloroform (50 ml) and washed with 10% HCl followed by a water wash. Theorganic phase was dried over anhydrous sodium sulfate to obtain whitesolid of 1,4-dipalmitoyl-tartarate-2,3-disuccinic acid or DPTSA (2.45gm, 94% yield, melting point 65° C.). ¹H NMR (300 MHz, CDCl₃): δ 5.72(s, 2H), 4.14 (t, 4H, CH₂, J=6.0), 2.80-2.65 (m, 4H, succinyl),1.67-1.58 (m, 4H, CH₂), 1.32-1.20 (m, 52H, CH₂), 0.88 (t, 6H, CH₃,J=6.4). ESI HRMS calculated for C₄₄H₇₈NaO₁₂(M⁺+Na) 821.54. Found 821.40.

1,4-Dipalmitoyl-tartarate-2,3-diglutaric acid (DPTGA, FIG. 3): Glutaricanhydride (2.61 gm, 22.7 mmol) and 1,4-dipalmitoyl tartarate (6 gm, 9.17mmol) in pyridine (15 ml) were heated at 80° C. for 2 h. The compound,1,4-dipalmitoyl-tartarate-2,3-diglutaricacid (DPTGA) was extracted intochloroform (100 ml) and washed with 10% HCl (50 ml) followed by a washwith water (50 ml). The organic phase was separated, dried over Na₂SO₄to obtain white solid (7.76 gm, 96% yield). ¹H NMR (300 MHz, CDCl₃): δ5.69 (s, 2H), 4.20-4.05 (m, 4H), 2.60-2.40 (m, 8H), 2.05-1.95 (m, 4H),1.65-1.55 (m, 4H), 1.40-1.10 (m, 52H), 0.87 (t, 6H, CH₃).

1,4-Disteroyl-tartarate-2,3-disuccinic acid (DSTSA): DSTSA wassynthesized following the scheme similar to that used for DPTSA, exceptdistearoyl tartaric acid ester (DSTA) was used as the starting lipid.DSTA was obtained commercially from VWR Scientific. DSTSA was collectedas white solid (93% yield, melting point 81° C.). ¹H NMR (300 MHz,CDCl₃): δ 5.75 (s, 2H), 4.20-4.05 (m, 4H, CH₂), 2.80-2.55 (m, 4H,succinyl), 1.70-1.55 (m, 4H, CH₂), 1.45-1.15 (m, 60H, CH₂), 0.88 (t, 6H,CH₃).

Cholesteryl hemisuccinate (CHEMS, FIG. 4): Succinic anhydride (1.55 gm,15.5 mmol) was added to a solution of cholesterol (5 gm, 12.9 mmol) inpyridine (10 ml). After heating at 80° C. for 3 h, the reaction mixturewas diluted with DCM and the organic phase was washed with 10% HCl (50ml) followed by a water wash (50 ml). The organic phase was separated,dried over Na₂SO₄ to obtain cholesterol hemisuccinate as white solid.The solid was recrystallized from DCM and hexane (5.78 gm, 92% yield,and melting point 176-178° C.). ¹H NMR (300 MHz, CDCl₃): δ 5.47 (d,vinylic-H, J=5.0), 4.70-4.60 (m, 1H), 2.75-2.65 (m, 4H), 2.38-2.35 (d,7H), 2.20-1.80 (m, 12H), 1.60-1.40 (m, 12H), 0.78-0.75 (m, 3H).

Isolation of Stroma-Free Hemoglobin

Concentrated stroma-free hemoglobin was isolated from the outdated redblood cell (RBC) units using a previously described method [Sakai etal., 1993]. Outdated RBC units were obtained from Sylvan Goldman Center,Oklahoma Blood Institute (Oklahoma City, Okla.). To enhance thestability of hemoglobin during the isolation process, the RBC suspensionwas purged with 0.22 μm-filtered carbon monoxide gas (CO) which convertshemoglobin into a relatively more stable carbonylhemoglobin (CO-Hb). Thecarbonylated RBCs (20 g/dl, 300 ml) were mixed with dichloromethane(DCM, 60 ml) and mixed for 10 min. The precipitate was allowed tosettle, and the supernatant was again extracted with DCM. The extractionwas repeated 3 times to remove all the lipid material soluble in organicphase. The residual DCM in the aqueous phase containing CO-Hb solutionwas removed under vacuum in an R-210 rotavapor (Buchi Corporation, NewCastle, Del.) at 40° C. in dark. The resultant CO-Hb solution wasfurther heated at 60° C. for 1 hour to denature and precipitate anymethemoglobin in the solution. The precipitate was removed bycentrifugation at 8000 rpm for 20 min at 4° C. in a Sorvall RC-5Brefrigerated superspeed centrifuge. The supernatant was purified fromfine remnants of particulate matter by sequentially passing it throughtangential-flow FiberFlow capsules having 100 and 50 nm cut-off(Minntech, Minneapolis, Minn.). Finally, the purified hemoglobinsolution was concentrated to about 38 g/dl using a 30 kDa cut-offPrep/Scale-TFF filter cartridge (Millipore, Billerica, Mass.). The finalpreparation was characterized for oxygen affinity, CO-Hb content, MetHbconcentration and endotoxin.

Preparation of Liposome-Encapsulated Hemoglobin (LEH)

The LEH was prepared in several steps described as follows:

Preparation of pro-liposomes: Freeze-thaw (FT) method was used for thepreparation of pro-liposomes.1,2-disteroyl-sn-glycero-3-phosphatidylcholine (DSPC), cholesterol(CHO), anionic lipid,1,2-disteroyl-sn-glycero-3-phosphoethanolamine-N-[monomethoxypoly(ethylene glycol) (5000)] (DSPE-PEG₅₀₀₀) and vitamin E in38.85:38.85:20:0.3:2 mol % were dissolved in a mixture ofchloroform:methanol (2:1) and transferred to a round bottom flask. Thesolvent mixture was evaporated at 58° C. on rotavapor to form a thinfilm. Any trace of organic solvent was removed by keeping the film undervacuum for 12 h. The phospholipid film was rehydrated with sodiumhydroxide solution equimolar to the carboxyl groups present in theanionic lipid to maintain total lipid concentration to 12 mM. The pH ofthe lipid suspension was adjusted to 6.8-7.0. This suspension of largemultilamellar vesicles was subjected to 8 FT cycles. An FT cycleconsisted of snap-freezing the suspension in liquid nitrogen followed byimmediate thawing in a 58° C. water bath. DPPC-based pro-liposomes wereprepared using DPPC, CHO, anionic lipid, DSPE-PEG₅₀₀₀ and vitamin E in38.85:38.85:20:0.3:2 mol % and processed as above except that thethawing was carried out at 42° C. After the final FT cycle, thepro-liposome suspension was shell-frozen in a lyophilization flask andsubjected to a 48 h lyophilization cycle in a Triad Lyophilizer(Labconco, Kansas City, Mo.). The dried pro-liposome material was storedat −20° C. until further used.

Optimization of hemoglobin encapsulation: The effect of synthesizedanionic lipids on the efficacy of hemoglobin encapsulation was examined.The lyophilized DPPC-based pro-liposomes containing 20 mol % of theanionic lipid were allowed to warm up to 25° C., and the powder wasaseptically and gradually added to a highly concentrated, purified 5 mlHb solution (38 g/dl, <0.25 endotoxin units/ml) with constant stirring.The entire process was carried out under HEPA-filtered laminar flowhood; during entire processing, the temperature was maintained to 25° C.After 2 hours of incubation the suspension was ultracentrifuged at35,000 rpm at 5° C. for 30 min in an Optima L-100 XP ultracentrifuge(Beckman Coulter, Fullerton, Calif.). The supernatant was removed andthe pellet was resuspended in phosphate-buffered saline (PBS, pH 7.4).The centrifugation cycle was repeated two times to completely get rid offree hemoglobin. Finally the pellet was resuspended in 1.0 ml of PBS (pH7.4) and the amount of encapsulated hemoglobin was determined bymonitoring the absorbance of LEH lysate in OBG at 540 nm [Tomita et al.,1968]. In order to determine the effect of increasing mol % of anioniclipid on hemoglobin encapsulation, three batches of DPPC-basedformulation were tested with 20, 28 and 40 mol % of CHHDA, andencapsulated Hb in respective formulations was estimated [Tomita et al.,1968].

Optimization of homogenization conditions: The homogenization processwas optimized separately using pro-liposome batches of identicalcomposition, except that no hemoglobin was introduced in thesepreparations. The particle size of LEH was reduced by high pressurehomogenization using Emulsiflex-C3 (Avestin Inc., Ottawa, Ontario,Canada). Parameters, such as number of homogenization cycles andpressure were optimized to obtain the LEH of size 200-300 nm.

Preparation of LEH: In order to validate the optimized lipid compositionand production protocol, two small LEH batches (LEH 1 and LEH 2, 25 mleach) were manufactured. The composition chosen was DPPC, CHO, CHHDA,DSPE-PEG₅₀₀₀, vitamin E in 34.85:34.85:28:0.3:2 mol %. In batch LEH 2,the p50 of hemoglobin was adjusted by adding pyridoxal-5′-phosphate(PLP, 2.5 molar times of hemoglobin), to the carbonylhemoglobin solutionand this solution was used for rehydration of lyophilized pro-liposomes.No PLP was used in LEH 1. Strict aseptic conditions were maintainedthroughout the preparation in a laminar flow hood. Final size reductionof hemoglobin containing pro-liposomes was carried out by high pressurehomogenization in Emulsiflex-C3 at 20K psi for 4 cycles—each cycleseparated by at least 30 minutes. The processing temperature wasmaintained at about 20° C. by immersing the heat transfer coil in anice-cold water-bath. Further processing of LEH was performed accordingto the method earlier published [Awasthi et al., 2004; Awasthi et al.,2004A]. Briefly, the free hemoglobin was separated from encapsulatedhemoglobin by tangential-flow filtration through 50 nm hollow fiberfilter using PBS (pH 7.4) as the diluting solvent. The purified LEH waspost-inserted with PEG-lipid by mixing an aqueous solution ofDSPE-PEG₅₀₀₀ with a dilute dispersion of LEH, such that the PEG-lipidconcentration remains below its critical micelle concentration. Toconvert encapsulated carbonylhemoglobin into oxyhemoglobin, thePEGylated LEH was exposed to a bright visible light from a 500W halogenlamp under saturating oxygen atmosphere at 4-8° C. The conversion wasmonitored spectrophotometrically. The dilute oxygenated LEH was furthersubjected to tangential-flow filtration (50 nm filter, PBS wash-fluid)to eliminate remnants of free hemoglobin. Finally, the LEH wasconcentrated to the desired batch volume and stored at 4° C.

Characterization of LEH

To visually document the formation of liposomes using the anionicnon-phospholipid, electron microscopy was performed at the University ofOklahoma at Norman (OK). The LEH preparations were characterized forhemoglobin content, methemoglobin, size, oxygen affinity, and lipidconcentration. Hb encapsulation was estimated by monitoring theabsorbance of the OBG lysate of LEH at 540 nm [Tomita et al., 1968]. Thephospholipid concentration was determined by Stewart assay [Stewart,1980]. Methemoglobin content was also measured [Matsuoka, 1997]. Oxygenaffinity (p50) was measured in a Hemox-analyzer (TCS Scientific, NewHope, Pa.). Briefly, a 50 μl sample of LEH was dispersed in a 4 mlphosphate buffer (pH 7.4) containing albumin and an antifoaming agent.The mixture was incubated at 37° C. for 5 minutes and aspirated into thecuvette for dual wavelength spectrophotometry. The sample was purgedwith N₂ gas to PO₂<2.0 mm Hg, before allowing air to attain PO₂>140 mmHg. The results were analyzed by HAS software provided with theinstrument. The particle size was determined by photon correlationspectroscopy using a Brookhaven particle size analyzer equipped withMass Option software. Zeta potential of preparations was measured in aZeta PLUS Zeta potential analyzer (Brookhaven Instruments Corp,Holtsville, N.Y.). Zeta potential of LEH was estimated, both, before(non-PEGylated LEH) as well as after (PEGylated LEH) PEG post-insertionand the values were compared to zeta potential of empty liposomescontaining 28% CHHDA or 20% DMPG. Preparations equivalent to about 40 μgof phospholipid in 1.5 ml of 0.22 μm filtered de-ionized water werescanned at 25° C. for 10 runs, each run consisting of 20 cycles. Zetapotential values were obtained as millivolt±standard error of mean.

Cell Culture and Cytotoxicity Studies

As a long-circulating oxygen carrier, LEH is expected to mostly interactwith two cell types—endothelial cells in the vasculature and macrophagesresponsible for clearing particulate material. Human Umbilical VeinEndothelial Cells (HUVEC, ATCC, Manassas, Va.) were grown in M-199medium supplemented with 10% (v/v) plain FBS, endothelial cell grownsupplement (ECGS) (20 μg/ml), heparin (90 mg/L), penicillin G (100 U/ml)and streptomycin (100 μg/ml) (Sigma-Aldrich, St. Louis, Mo.). Cells weremaintained at 37° C. in 5% CO₂ atmosphere. Cells were used after 3passes for cytotoxicity study. RAW 264.7 cells (ATCC, Manassas, Va.)were grown in Dulbecco's modified Eagle's medium supplemented with 10%FBS (ATCC) at 37° C. in 5% CO₂ atmosphere. Cells were grown in 96-wellplates at a density of 10⁴ cells/well. After 24 h of culture, the cellswere treated with 100 μl of either, 1, 2 or 5 mg/ml of PEGylated ornon-PEGylated LEH. Separately, cytotoxicity of empty liposomes carrying10, 20 and 28 mol % of CHHDA was also evaluated (5 mg/ml, 100 μl). Emptyliposomes containing 0 mol % CHHDA and those containing DMPG at 20 mol %were taken as controls for comparison. The cytotoxicity was measured asthe decrease in hexosaminidase activity in cells after 24 h as describedby Landegren [Landegren, 1984].Para-nitrophenol-N-acetyl-beta-D-glucosaminide was used as the substratefor hexosaminidase enzyme.

Platelet Activation by LEH In Vitro

Fresh human whole blood (5 ml) was obtained from a normal healthyvolunteer. The blood was anticoagulated by acid-citrate-dextrosesolution (9 blood:1 ACD, volume ratio). Platelet rich plasma (PRP) wasseparated from other cellular components by centrifugation at 150 g for8 min. The aliquots of PRP (about 0.5 million platelets) were treatedwith 5 mg/ml of lipid as LEH. Two preparations of LEH were tested—onewith PEG-coating and the other without PEG-coating. Adenosinediphosphate (ADP, 100 μM) was used as the positive control. After 5 and30 min of incubation The samples were labeled with anti-human plateletantibodies for flow cytometry [Shigeta et al., 2003].

Murine antihuman monoclonal fluorescein isothiocyanate (FITC)-conjugatedCD61 was used to identify all platelets. Murine antihuman monoclonalphycoerythrin (PE)-conjugated CD62P was used to detect plateletactivation. Isotype controls were run in parallel with all monoclonalantibodies: FITC-conjugated immunoglobulin G1 and PE-conjugatedimmunoglobulin G1. All antibodies and isotype controls were obtainedfrom eBioscience (San Diego, Calif.). Briefly, about 10 μl of 1:10diluted PRP was incubated in the dark at room temperature with a mixtureof 20 μL of FITC-conjugated CD61 antibody and 20 μl of PE-conjugatedCD62 antibody (each equal to about 0.25 μg and 0.125 μg antibodies,respectively). After 20 minutes, the platelets were thrice washed withPBS, followed by overnight fixing with 1 mL of ice-cold 1%paraformaldehyde in dark at 4° C. The samples were subjected to flowcytometric analysis in the Core Facility at the OUHSC on a FACS Calibur(BD and Company, Franklin Lakes, N.J.) equipped with an argon ion laserand CellQuest software (BD and Company, Franklin Lakes, N.J.). Forwardlight scatter and 2 fluorescent signals were determined for each cell,and at least 10000 platelet events were collected. The plateletpopulation was logic gated by a forward scatter versus side scatter dotplot gate and by an FITC-conjugated CD61 versus side scatter dot plotgate. CD62P-positive activated platelets were identified by fluoresceinintensity greater than that of the appropriate isotype control stainingsample. The frequencies of CD62P-positive platelets were expressed aspercentages of the total platelet population [Shigeta et al., 2003].

Data Analysis:

Data are presented as mean±SD. The statistical differences weredetermined by the paired Student t test for P<0.05.

Results of Example 3

Several novel anionic non-phospholipids were synthesized to enableenhanced encapsulation of hemoglobin inside the liposomes. FIG. 1-4 showthe synthetic scheme for various anionic non-phospholipids. Theinventors' observations in the development of an optimized and improvedLEH formulation based on these anionic non-phospholipids are recorded infollowing paragraphs.

The reaction yields for CHHDA were routinely above 75%. CHHDA wascharacterized by proton NMR and high resolution mass spectrometry(HRMS). The diagnostic spectroscopic data described below confirmed thesuccessful synthesis of CHHDA. ESI-HRMS showed molecular ion peak at538.40 and ¹H NMR showed the disappearance of vinylic protons at δ5.80-5.20 ppm. The lipid peak intensity at δ 1.35-1.15 ppm was increasedindicative of the hexadecylamine esterification. In DSC, CHHDA lipidshowed endothermic events around 50° C., prior to the melting at around80° C. (FIG. 5). Noticeable exothermic events were observed around roomtemperature, and the lipid showed two melting events. The peaktemperatures on the graph are printed by the instrument software. Thesemay not be melting points, but are the temperatures corresponding to themaximum of the exothermic release of heat.

DPTGA formation was confirmed by the appearance of glutaric protons at δ4.20-4.05, lipid CH₂ protons at 2.05-1.95 and 1.40-1.10 ppm asmultiplets in ¹H NMR. In case of DSTSA, the protons at the site ofesterification shifted downfield to δ 5.69 from 4.52 ppm suggestingesterification. The stearoyl tartarate protons on —OH bearing carbonsappeared as broad doublets at 4.50 ppm and shifted downfield to 5.75 assinglet after esterification of —OH groups. The succinyl protonsappeared at 2.55-2.80 ppm as multiplets in ¹H NMR spectrum. CHEMS showedmelting point of 176-178° C. where as cholesterol has a melting point of148-150° C. The succinyl protons of CHEMS appeared at δ 2.75-2.60 in ¹HNMR.

The freeze-thaw method was used to prepare DSPC and DPPC basedpro-liposomes, before encapsulation of hemoglobin. The particle size ofDSPC-based pro-liposomes was reduced to 397.4±6 nm after 8 FT cycles(Table 1). When sonication was used along with thawing, thepro-liposomes were reduced to an undesirably small size (210-215 nm). Incase of DPPC-based composition, pro-liposomes of size 441.0±18 nm wereproduced after only 4 FT cycles. The effect of bath-sonication duringthawing was similar to that seen with DSPC pro-liposomes, and the finalsize of DPPC pro-liposomes was about 213±2 nm.

To investigate the effect of anionic lipids on hemoglobin encapsulation,DPPC based pro-liposome containing 20 mol % of one of the synthesizedanionic lipids were incubated with carbonylhemoglobin solution. Thehemoglobin content of CHHDA-containing liposomes was found to be thehighest (3.15 g/dl) whereas the hemoglobin content of DPTGA containingliposomes was the lowest (1.55 g/dl) (Table 2). The hemoglobin-to-lipidratio for CHHDA liposomes was 1.2. Methemoglobin formation in case ofall the anionic lipids except CHEMS was less than 2%. For unknown andunexplored reasons, CHEMS tended to induce methemoglobin formation tothe extent of 40% of total hemoglobin. The visible appearance of thispreparation was dark brown whereas all other preparations were shinydeep red in color. Further, pro-liposomes containing increasing amountsof CHHDA were prepared to investigate the effect on Hb encapsulation.The Hb encapsulation was found to increase with increasing CHHDA mol %to a certain limit (FIG. 6). The highest encapsulation (4.02 g/dl) wasobserved with 28 mol % of CHHDA. At 40 mol %, CHHDA appeared to disruptliposome structure as no LEH pellet was observed afterultracentrifugation. As maximum Hb encapsulation was with 28 mol %CHHDA, effect of 28 mol % CHHDA on the bilayer structure of liposomeswas investigated by scanning electron microscopy. As shown in FIG. 7,electron micrographs of LEH showed typical structure of liposomes. Insummary, CHHDA up to 28% can be used as an anionic lipid component inLEH preparations.

In a separate set of experiments, the homogenization process wasoptimized for pressure and number of passes. At 15K psi pressure,homogenization for 4 or 5 passes did not reduce the particle size below400 nm (Table 3). Increasing the pressure to 20K psi produced theliposomes in 200 nm range. As the desired size range was achieved at acombination of 4 passes at 20K psi, this set of conditions was selectedfor scale up of manufacturing further batches of LEH. Two batches of LEHwere prepared—one containing PLP and the other without PLP. LEH 1 andLEH 2 had particle size of 273.4±4.2 and 222.6±1.3 nm, respectively. LEH1 and LEH 2 were characterized as described in Table 4. Both the batcheshad a high hemoglobin content and hemoglobin-to-lipid ratio. Bothpreparations had less than 2% methemoglobin. The entire LEH processinghas been schematized in FIG. 8. Separately the possibility of CHHDAhaving any effect on the oxygen affinity of hemoglobin was investigated(Table 5). It is clear that even at 28 mole %, CHHDA does not have anysignificant impact on p50 value on its own. On the other hand, theaddition of PLP to hemoglobin prior to encapsulation altered the p50 ofthe final product from about 19 to 33 Torr (Table 4). The p50 value ofLEH 1 without PLP was 18.94, whereas LEH 2 with PLP had p50 value of33.14 Torr.

TABLE 1 Sizes of Pro-liposomes after Freeze-thaw Phospholipid FT cycleSonication? Size (nm) DSPC 4 —  639.4 ± 13 DSPC 4 Yes 215.1 ± 2 DSPC 8 —397.4 ± 6 DSPC 8 Yes 210.3 ± 3 DPPC 4 —   441 ± 18 DPPC 4 Yes 213.2 ± 2

TABLE 2 Effect of various anionic lipids on hemoglobin encapsulationPhospholipid Mole % Hb content (g/dL) Hb/lipid DPTA 20 2.6 1.26 DPTGA 201.55 1.2 DSTSA 20 2.09 1.7 CDHHA 20 3.15 1.2 CHEMS 20 Hb oxidation —

TABLE 3 Optimization of homogenization process Number of Pressure Sr. NoPasses (psi) Size (nm) 1 4 15K 405.1 ± 30 2 5 15K 435.3 ± 16 3 5 20K 199.1 ± 2.8 4 4 20K  222.6 ± 1.3 5 3 22K 263.7 ± 4 

TABLE 4 Characteristics of LEH Batches Parameter LEH 1 LEH 2 Size (nm)273.4 ± 4.2 222.6 ± 1.3 Hb (g/dl) 4.45 4.19 Hb/Lipid ratio 1.2 1.16 p50(Torr) 18.94 33.14 Met-Hb (%) <2 <2

TABLE 5 Effect of CDHHA on p50 of hemoglobin Sr. No. TDSA-H (mol %) p50(Torr) 1 0 9.94 2 20 10.18 3 28 10.85

Under the conditions of measurement, the zeta potentials of empty 20 mol% DMPG liposomes and empty 28% CHHDA liposomes were found to be−90.91±2.62 and −74.67±0.65 mV, respectively. Clearly, even at 28%concentration, the apparent negative potential of the CHHDA liposomeparticle was significantly lower than that of liposomes containing about⅔^(rd) the amount of DMPG. The liposome's surface charge is governed byhow the negatively-charged constituent is packed in the liposomestructure, and how ionized the constituent becomes under measuringconditions. In comparison, the non-PEGylated LEH showed zeta potentialvalue of −33.86±1.0. Presence of hemoglobin seemed to reduce negativepotential of the liposome particles, possibly by interacting andneutralizing the negative charge of the liposome bilayer. PEGylation ofLEH further reduced zeta potential to −27.98±2.4 mV. PEG coating ofparticles is known to decrease the charge effect under in vivoconditions.

As a long-circulating oxygen carrier, LEH is expected to mostly interactwith two cell types—endothelial cells in the vasculature and macrophagesresponsible for clearing particulate material. The toxicity of the LEHformulation was investigated in HUVEC and RAW cell lines. Cells weregrown in 96-well plates at a density of 10⁴ cells/well. After 24 h ofculture, the cells were treated with LEH and empty liposomes carryingvarying amounts of CHHDA or 20 mol % DMPG; the cytotoxicity was measuredas the decrease in hexosaminidase activity in cells after 24 h. As shownin FIG. 9, the LEH showed no toxicity in the HUVEC or RAW cells ascompared to controls (100%) at any concentration that was tested. Alsono significant difference was observed in cytotoxicity pro of PEGylatedand non-PEGylated liposome at the tested concentration (p>0.05). All theempty liposome formulations tested appeared to be non-toxic to the cells(p>0.05).

Previous studies have shown that liposome preparations, such as LEH havea tendency to activate platelets. A flow cytometry-based in vitro assaywas performed to investigate if the LEH treatment activates plateletsand whether PEGylation has any impact on this phenomenon. CD61 is amarker of platelet glycoprotein IIIa, which is found on both normal(resting) and activated platelets. On the other hand, CD62P is found onthe external membrane of activated platelets only. As shown in FIG. 10,LEH treatment did not demonstrate any platelet activation phenomenon.PEGylation of LEH had no effect on the LEH-mediated response of theplatelets. Treatment with ADP (100 μm) caused about 56% activation ofplatelets. The results were identical when the lipid-plateletinteraction was allowed to occur for 30 min (data not shown).

Discussion of Example 3

LEH is primarily mostly composed of a combination of saturatedhigh-carbon phospholipids, such as distearoyl phosphatidylcholine (DSPC,T_(m) 55° C.) and dipalmitoyl phosphatidylcholine (DPPC, T_(m) 41° C.)and cholesterol. Although, hemoglobin encapsulation in the liposomes hasnot been possible to match hemoglobin content of RBCs, it is desirableto encapsulate large amounts of hemoglobin within a minimum amount oflipid. Hemoglobin interacts with the phospholipid bilayer by bothhydrophobic and ionic forces [Szebeni et al., 1988]. Ionic interactionseems stronger than the hydrophobic interaction and is dependent on pHand ionic strength of the medium [Pitcher et al., 2002]. As such,negatively charged lipids enhance encapsulation by interacting withoppositely charged domains of proteins. Using about 9 mol % of DPPG, inconjunction with optimal encapsulating conditions, Tsuchida andcoworkers achieved the Hemoglobin-to-lipid ratio of 1.61 [Sakai et al.,1996; Takeoka et al., 1996]. Below its isoelectric point, Hb carries apositive charge and electrostatically interacts with negatively chargelipids resulting in increased encapsulation [Sakai et al., 1996].

Apart from the beneficial interaction with hemoglobin, anionic lipidsare known to undesirably enhance interaction of liposomes withcomplement and other opsonizing proteins in vivo [Miller et al., 1998;Szebeni, 1998; Semple et al., 1998]. Such interactions result in a rapiduptake of LEH by the RES, and toxic effects manifested asvasoconstriction, pulmonary hypertension, dyspnea, etc. Anionicphospholipids also enhance the rate of hemoglobin oxidation and displaceheme relative to globin [Szebeni et al., 1988]. It is possible topartially reduce the toxicity of anionic phospholipids by PEGmodification of LEH surface [Awasthi et al., 2004A]. It is believed thata hydrophilic PEG coating on the liposome surface creates a stericbarrier, enabling liposomes to circulate longer [Torchilin et al.,1994]. Recently, the inventors have shown that PEGylation significantlyreduces the thrombocytopenic effect of LEH administration in a rabbitmodel [Awasthi et al., 2007]. In view of the drawbacks of anionicphospholipids, an amino acid-based synthetic anionic lipid,1,5-dipalmitoyl-1-glutamate-N-succinic acid, has been used in LEH [Souet al., 2003]. It is believed that this amino acid-based lipid is bettertolerated than anionic phospholipids.

The overall object of this work was to develop an LEH formulation withhigh Hb encapsulation based on ionic interaction with members ofliposomal bilayer. In order to achieve this objective, several anioniclipids without phosphate groups were synthesized. Four structurallydifferent anionic lipids, as well as a cholesterol hemisuccinate(CHEMS), were synthesized for this purpose. All the anionic lipids weresynthesized keeping in mind that they will be incorporated as acomponent of liposome membrane. All the lipids have features normallyobserved in phospholipid and considered essential for stable liposomeformation, except that they do not have a phosphate group. These includea polar head group, at least a 16-member carbon double chain and meltingpoints and molecular weights in the range of commonly used phospholipid.Negative charge was imparted by having either one (CHHDA,) or two (DTPA,DSTSA, DPGA and CHEMS) —COOH groups on the polar head.

The first step in LEH production was to make either DSPC or DPPC basedpro-liposomes containing the novel anionic lipids. FT method effectivelyproduced pro-liposomes in the size range of 397-441 nm for DSPC and DPPCbased pro-liposomes, respectively. FT cycles reduce the lamellarity ofliposomes thus converting multilamellar vesicles into unilamellarvesicles. Reduction in lamellarity is helpful in increasingencapsulation efficiency on lipid basis. During freezing process icecrystals are formed and disrupt the bilayers. Disrupted bilayersreassemble to form newer vesicles. Thus FT increases population ofvesicles while decreasing the lamellarity [Sou et al., 2003;Sriwongsitanont, 2004]. When FT process was coupled with sonicationduring thawing, the size reduction reached undesirably lower values.Hence the use of sonication was discontinued during furtherdevelopmental stages. As expected, the number of cycles required toattain optimal pro-liposome size was dependent on the phospholipidused-DPPC (Tm=41° C.) pro-liposomes took fewer cycles than DSPC (Tm=55°C.) pro-liposomes. DPPC based formulation was considered more suitablefor protein encapsulation due to milder processing conditions required.Hence this formulation was persisted with during further experiments.

The DPPC-based pro-liposomes were used to encapsulate hemoglobin. It wasobserved that liposomes containing CHHDA had the highest encapsulationefficiency. The experience with CHEMS was poor. Cholesterol is anessential component of most of the liposomes and is used sometimes ashigh as 50 mol %. An attempt was made to examine if some or all of thecholesterol can be replaced with CHEMS in LEH. It was believed thatdifference in the spatial orientation of CHEMS compared to anioniclipids in bilayers was responsible for the poor encapsulation. Somehow,CHEMS-containing LEH demonstrated high Met-Hb formation. The exact causeof superior performance of CHHDA is not known at this stage. CHHDA hasonly one —COOH group whereas other anionic lipids have two. This clearlyindicates that the encapsulation is not a function of number of —COOHgroups only. Orientation of lipids within the bilayer might affectpacking of liposomes and rigidity. In this context it is worthwhile tonote the inventors' observation that all preparations except thosecontaining CHHDA revealed swelling-like phenomenon to varying degrees.Swelling did not increase with number of FT cycles and particle size wasin the desired range of 400-450 nm immediately after FT. Howeverpro-liposomes (except CHHDA) were observed to undergo gel formationafter 24 hr which was an indication of vesicle fusion. This might havesome relation to the low encapsulation efficiency observed with theselipids. Since CHHDA resulted in maximum Hb encapsulation, furtheroptimization was carried out using this anionic lipid. As the highest Hbencapsulation (4.02 g/dl, Hb/lipid ratio 1.2) was observed at 28 mol %of CHHDA, LEH 1 and LEH 2 were prepared with 28 mol % of CHHDA. At 40mol % of CHHDA, liposomes did not form. High mol % of CHHDA might haveresulted in a very high electrostatic repulsion and disruption ofbilayers.

The oxygen affinity is measured in terms of the partial pressure ofoxygen required to saturate 50% of hemoglobin or p50. p50 of hemoglobinis altered by several allosteric modifiers. Since anionic lipidsinteract with hemoglobin fairly intimately, it was interesting to studyif CHHDA at 28 mol % concentration influences p50 of Hb. There wasinsignificant effect of CHHDA on the p50 of encapsulated hemoglobin.More importantly, interaction between CHHDA and Hb did not affect theability of PLP to enhance p50. PLP is an established allosteric modifierand its use in LEH2 easily altered p50 to 33 Torr. The inventors'previous experience suggested that LEH made up of the purifiedstroma-free hemoglobin (SFH) should have shown lower p50 value (<10Torr). However, in case of LEH 1, the batch without PLP, the p50 valuewas about 19. The reasons for this discrepancy are not known, but aresuggestive of some role played by the age of the outdated RBCs, state oftheir storage and the method of isolation. The previously reportedpurified SFH with high oxygen affinity was isolated from outdated RBCskept frozen for several months; the isolation technique was based onhypotonic lysis of RBCs followed by sequential filtration throughfilters of decreasing pore size [Awasthi et al., 2004; Awasthi et al.,2004A]. In comparison, the SFH used in this work was from RBCs collectedwithin 1 week of being outdated, kept at 4° C., and hemoglobin isolatedby method based on the use of DCM [Sakai et al., 1993].

Using high-pressure homogenization, the inventors were able to reducethe particle size of LEH to a desired range. It has been reportedearlier that the optimum size of PEGylated LEH for prolonged circulationafter administration is 210-240 nm [Awasthi et al., 2004A].

As a long-circulating oxygen carrier, LEH is expected to mostly interactwith two cell types—endothelial cells in the vasculature and macrophagesresponsible for clearing particulate material. The toxicity of the LEHformulation was studied in HUVEC and RAW cell lines. It was found thatthe presence of CHHDA in LEH is not toxic to the endothelial cells andmacrophages in culture.

In addition, the LEH preparations containing CHHDA were not found toactivate platelets in vitro. Studies in animal models receiving LEH orliposomes noted a formulation-dependent transient decrease incirculating platelets immediately following an intravenous infusion[Rabinovici et al., 1994]. Such an undesired reaction remains anobstacle to the successful application of LEH as a universalresuscitation fluid. The observation that in vitro platelet aggregationis not affected by incubation with LEH indicates an essential role of asystemic element in inducing platelet reaction [Phillips et al., 1997].It has been since demonstrated that complement system plays an integralrole in LEH-induced thrombocytopenia [Goins et al., 1997], causingactivation of classical complement pathway after interaction between thephospholipid bilayer and C reactive protein. A recent article has alsoshown that anionic charge on the surface of the liposome plays a keyrole in activation of both classical and alternative complement pathway[Moghimi et al., 2006]. Liposomes containing negatively-chargedphospholipids induce a very rapid decline in circulating platelets thatis more severe, and that takes a longer time to recover to normal levelsas compared to the liposomes consisting of neutral phospholipids.Liposomes containing phosphatidylglycerol form micro-aggregates withplatelets in vitro, and it has been suggested that the sequestration ofthese micro-aggregates by the reticuloendothelial system causes thedecline in circulating platelets[Loughrey et al., 1990].

The utility of LEH is most obvious when there is reduction in the oxygencarrying capacity secondary to a severe blood loss. Uncontrolledhemorrhage is also characterized by progressive depletion of circulatingthrombocytes resulting in an inability to initiate effective hemostasis.In these circumstances, a further thrombocytopenic reaction toresuscitative fluid would exacerbate an already compromised hemostasiscondition. Therefore, the LEH preparation should be formulated toeliminate the platelet reaction seen with liposome administration. Theinventors recently reported the effect of charge and PEGylation onplatelet activation by LEH in a rabbit model [Awasthi et al., 2007]. Thetested LEH preparation contained dimyristoylphosphatidylglycerol (DMPG)as the anionic lipid.

Therefore, a formulation of LEH containing novel anionicnon-phospholipid has been shown herein to enhance hemoglobinencapsulation. The LEH formulation was not toxic to endothelial cellsand macrophages, and did not demonstrate any platelet activatingtendency.

Example 4

Effectiveness of LEH resuscitation in a rat model of hemorrhagic shock.

The effectiveness of LEH on cerebral energy metabolism was evaluated ina 40% hemorrhagic shock model in rats using proton and phosphorousmagnetic resonance spectroscopy (¹H-MRS and ³¹P-MRS). The study lastedabout 6 h, and animals infused with LEH survived the study duration. Thestudy design is shown in FIG. 11. ¹H-MRS data showed that the levels ofmarkers of anaerobic metabolism, like lactate and pyruvate, increasedpost-bleeding but recovered closer to baseline levels afterresuscitation with LEH. N-Acetylaspartate (NAA) also followed a similartrend. NAA is almost exclusively present in neurons, and its lower thannormal levels indicate neuronal death. The fact that NAA levels returnto the baseline after LEH resuscitation indicate the protectiveinfluence of LEH on neurons. The information obtained after ³¹P MRS alsoendorse the efficacy of LEH in oxygen delivery. The levels of PCr fallpost-bleeding, which is an indication of ATP production at the expenseof PCr via the creatine kinase pathway. Increase in Pi levels postbleeding suggest ATP breakdown. Both these markers return to theirbaseline levels after LEH resuscitation. The changes in ATP levels weremonitored by following the β-ATP peak, as γ-ATP and α-ATP peaks containsome contribution from β and α-ADP phosphates. The β-ATP level fallsafter bleeding (correlates well with post-bleed Pi increment) andrecovers after LEH resuscitation. The trend followed by the metabolitesdescribed above is clearly indicative of a change of aerobic energymetabolism to an anaerobic pathway after bleeding and its return toaerobic pathway after LEH resuscitation. The observations thathemorrhage results in the appearance of a lactate peak in ¹H-MRS andthat the lactate peak disappears after LEH resuscitation demonstratethat LEH resuscitation is able to change post-bleeding anaerobicmetabolism in the brain to an aerobic one. Also it can be concluded that¹H and ³¹P MRS are effective tools to study this alteration in thepattern of energy metabolism.

Thus, in accordance with the present invention, there have been providedanionic lipid and liposome/lipid nanostructures, as well as methods ofproducing and using same, that fully satisfy the objectives andadvantages set forth hereinabove. Although the invention has beendescribed in conjunction with the specific drawings, experimentation,results and language set forth hereinabove, it is evident that manyalternatives, modifications, and variations will be apparent to thoseskilled in the art. Accordingly, it is intended to embrace all suchalternatives, modifications and variations that fall within the spiritand broad scope of the invention.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A method of producing a liposomal structurehaving at least one molecule encapsulated therein, the method comprisingthe steps of: forming a liposomal structure having at least one moleculeencapsulated therein, the liposomal structure formed of an anionicnon-phospholipid having the structure represented by the followinggeneral formula:

wherein R is NH; R′ is at least one of H, an alkyl group, Na, Li, K, ametal and a halogen; R″ is at least one of a —CH₂— group and a —CH₂CH₂—group; and n is an 8-16 carbon chain and x is an 8-16 carbon chain. 2.The method of claim 1, wherein the step of forming the liposomalstructure having the at least one molecule encapsulated therein isfurther defined as comprising the steps of: dispersing the anionicnon-phospholipid in an aqueous solution; lyophilizing the solution toform a proliposomal structure; and mixing the proliposomal structurewith at least one molecule in aqueous solution, whereby the at least onemolecule is encapsulated, thereby forming a liposomal structure havingat least one molecule encapsulated therein.
 3. The method of claim 1,wherein the anionic non-phospholipid is present in a range of from about1% to about 30% of the total lipid present in the liposomal structure,and wherein the liposomal structure comprises a particle size in a rangeof from about 50 nm to about 500 nm, and a volume average particle sizein a range of from about 10 nm³ to about 5,000 nm³.
 4. The method ofclaim 1, wherein the anionic non-phospholipid is 2-carboxyheptadecanoylheptadecylamide (CHHDA).
 5. The method of claim 2, wherein the liposomalstructure further comprises at least one phospholipid incorporatedwithin a liposomal membrane of the liposomal structure, the at least onephospholipid selected from the group consisting of phosphatidylcholine,phosphoethanolamine, phosphatidylglycerol, and wherein the step ofdispersing is further defined as dispersing the anionic non-phospholipidand the phospholipid in an aqueous solution, and wherein the at leastone phospholipid is present in a range of from about 30% to about 99% ofthe total lipid present in the liposomal structure.
 6. The method ofclaim 1, wherein the at least one molecule encapsulated therein isselected from the group consisting of a therapeutic molecule, areductant, an antioxidative enzyme, an oxygen-affinity modifier, anenzyme, a chelating agent, a pharmaceutical agent, antibiotic agents,antiviral agents, antifungal agents, chemotherapeutic agents,anti-inflammatory drugs, curcumin or a derivative thereof, diagnosticagents, radiodiagnostic and radiotherapeutic agents, and combinationsthereof.
 7. The method of claim 6, wherein the at least one moleculeencapsulated therein is at least one of hemoglobin and3,5-bis(2-fluorobenzylidene)-4-piperidone (EF24).
 8. A method,comprising the steps of: administering an effective amount of apharmaceutical composition to a patient in need thereof, thepharmaceutical composition comprising a liposomal structure comprising:an anionic non-phospholipid having the structure represented by thefollowing general formula [1]:

wherein R is NH; R′ is at least one of H, an alkyl group, Na, Li, K, ametal and a halogen; R″ is at least one of a —CH₂— group and a —CH₂CH₂—group; and n is an 8-16 carbon chain and x is an 8-16 carbon chain; andat least one molecule encapsulated therein.
 9. The method of claim 8,wherein the at least one molecule encapsulated in the liposomalstructure is hemoglobin.
 10. The method of claim 8, wherein the at leastone molecule encapsulated in the liposomal structure is3,5-bis(2-fluorobenzylidene)-4-piperidone (EF24).